[Fluorescent fusion proteins with 10th human fibronectin domain].

In the current paper we describe a new type of hybrid molecules including red fluorescent protein mCherry and 10th type III human fibronectin domain (10Fn3) - one of the alternative scaffold proteins which can be used for the construction of antibody mimics with various binding specificity. We have constructed different gene variants encoding for the hybrid fluorescent protein and studied their expression in Escherichia coli cells. It was shown that N-terminal position of mCherry and modification of its N-terminal amino acid sequence promotes efficientbacterial expression of the hybrid protein in the soluble form. On the basis of the proposed construction we have obtained the hybrid fluorescent protein ChIBF, containing alphaVbeta3-integrin binding vari- ant of 10Fn3, and demonstrated the possibility of its utilization for the visualization of alphaVbeta3-integrin at the surface of MDCK epithelial cells by confocal microscopy.

[Production and properties of human tumor necrosis factor peptide fragments].

The tumor necrosis factor (TNF) is a proinflammatory cytokine that plays a pivotal role in the regulation of the human immune system. Studies of the TNF functional topography are a challenging task in bioengineering. We have produced genes encoding the peptides Dl (3-30), D2 (31-85), D3 (86-114), and D4 (115-157), which correspond to isolated fragments of the informational structure of TNF. These genes were expressed in E. coli cells at a high level in a soluble form. We have shown that hybrid proteins SD2 and SD4 tend to form soluble aggregates, which can be destroyed by urea treatment. Purified peptides Dl, D3, and D4 possess a similar secondary structure with dominating beta-structural elements. The analysis of the biological activity of these peptides has shown that they do not exhibit cytotoxic properties on L929 murine fibroblasts. The simultaneous addition of Dl with full-length TNF results in the concentration dependent suppression of TNF activity.

[Recombinant human insulin. VII. Increased efficacy of chromatographic separation based on a principle of bifunctionality]. / Genno-inzhenernyi insulin cheloveka. VII. Povyshenie éffektivnosti khromatograficheskogo razdeleniia pri ispol'zovanii printsipa bifunktsional'nosti.

Retention mechanisms of insulin and deamido[AsnA21] insulin on the bifunctional sorbent Armsphere-C8(PR) in conditions of reversed-phase chromatography (HPLC and ion-pair HPLC) were studied. In accordance with the chemical differences of these proteins, molecular mechanisms of their interaction with silica gel modified with hydrophobic and ion-exchange groups were revealed. The possibility of simultaneous interaction of sorbed proteins with the stationary phase by both mechanisms under conditions of reversed-phase HPLC was demonstrated. The dependences of the separation selectivity and resolution on the mobile phase composition and properties (a salt buffer type, pH, ionic strength) were found. It was demonstrated that the separation selectivity can be regulated by altering the contribution of each of the two separation mechanisms and the bifunctional sorbent used allows higher selectivity in the separation of close protein analogs than monofunctional sorbents.

[An effective method of purifying recombinant human tumor necrosis factor alpha]. / Effektivnyi metod ochistki rekombinantnogo alpha-faktora nekroza opukholei cheloveka.

An efficient and productive isolation method for human recombinant tumor necrosis factor alpha from Escherichia coli cells was developed. The method includes a membrane filtration step, two steps of ion-exchange chromatography, and gel filtration on a Sephadex G-25 column. The target product was obtained with approximately 50% total yield and greater than 95% purity according to PAGE and HPLC.

[Effect of the topography of the signal peptidase site on the effectiveness of secretion of recombinant human granulocyte-macrophage colony-stimulating factor into Escherichia coli periplasm]. / Vliianie topografii saita signal'noi peptidazy na éffektivnost' sekretsii v periplazmu Escherichia coli rekombinantnogo granulotsitarno-makrofagal'nogo koloniestimuliruiushchego faktora cheloveka.

Synthesis of an artificial gene encoding the signal peptide of the Yersinia pestis capsule antigen (Caf1) was accomplished. A set of plasmids coding for hybrid proteins in which a modified sequence of the Caf1 signal peptide is connected to the amino acid sequence of the mature granulocyte-macrophage colony stimulating factor (GM-CSF) were constructed. Topography of the cleavage site of signal proteases was studied. The presence of an arginine residue within the N-terminal part of the mature human GM-CSF was shown to hinder the proper processing and translocation of the precursor through periplasmic membrane. A number of E. coli strains secreting biologically active mutants of human GM-CSF were obtained.

[Genetic engineering of human insulin. IV. Development and optimization of an analysis system using reversed phase high pressure liquid chromatography]. / Genno-inzhenernyi insulin cheloveka. IV. Razrabotka i optimizatsiia sistemy analiza s pomoshch'iu obrashchenno-fazovoi vysokoéffektivnoi zhidkostnoi khromatografii.

The effectiveness of the RP HPLC application for the step-by-step analysis of the recombinant insulin production was studied. Properties of a number of commercial and experimental columns in different chromatographic conditions were considered. A three-dimension optimization of selectivity and resolution versus pH and ion strength was carried out. A mechanism of the resolution and selectivity control is suggested.

[Genetically engineered human insulin. 1. HPLC in analyzing products of basic production stages]. / Genno-inzhenernyi insulin cheloveka. 1. VEZhKh v analize produktov osnovnykh stadii proizvodstva.

Application of some variants of HPLC for the step-by-step analysis of recombinant human insulin production was studied. Chromatographic columns with commercial and specially developed supports for size-exclusion, ion-exchange and reverse phase HPLC were used. Effective combinations of the chromatographic techniques for analysis of products and intermediates at every technological step were found and used for production of insulin. The authenticity of insulin obtained in the Shemyakin Institute of Bio-organic Chemistry by the scheme described in the present paper was confirmed by means of some physical and chemical methods and biological activity analysis.

[High performance liquid chromatography of nucleotides. Major methods and their development]. / Vysokoéffektivnaia zhidkostnaia khromatografiia nukleotidov. Osnovnye metody i ikh razvitie.

The separation of mono- and oligonucleotides possibilities by means of high performance ion-exchange, reversed-phase, so-called "ion-pair" and adsorption chromatography are studied. The influence of the eluent composition (solvent, salt) and pH on the retention, selectivity and resolution in reversed-phase and ion-exchange chromatography is investigated. The model of the hydrophobic-pair ion-exchange mechanism of ion-pair chromatography is considered. The conditions for analysis and preparative isolation of a desired component are optimized for selectivity, resolution and throughput. The methods for prediction of the optimal gradient elution program reasonable resolution at the desired retention time and for choosing the guard-column packing material are proposed. A design of the gradient for system and the version of slurry packing method for HPLC prolonged life-time columns are improved. The automatized analytical technique for determination of the oligonucleotide monomeric composition with two coupled microcolumns is described, that involves enzymatic digestion of an oligonucleotide followed by ion-exchange separation of the hydrolysate.